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Christopher M Dobson

Publications and source records attributed to Christopher M Dobson.

At least 19 recordsLinked to original sources
PubMed2006

Geometry, energetics, and dynamics of hydrogen bonds in proteins: structural information derived from NMR scalar couplings.

An accurate description of hydrogen bonds is essential to identify the determinants of protein stability and function as well as folding and misfolding behavior. We describe a method of using J couplings through hydrogen bonds as ensemble-averaged restraints in molecular dynamics simulations. Applications to the cases of ubiquitin and protein G show that these scalar couplings provide powerful structural information that, when used through the methodology that we present here, enables the description of the geometry and energetics of hydrogen bonds with an accuracy approaching that of high-resolution X-ray structures.

Computer Simulation↗
PubMed2006

Nature and significance of the interactions between amyloid fibrils and biological polyelectrolytes.

Charged polyelectrolytes such as glycosaminoglycans and nucleic acids have frequently been found associated with the proteinaceous deposits in the tissues of patients with amyloid diseases. We have investigated the nature and generality of this phenomenon by studying the ability of different polyanions, including DNA, ATP, heparin, and heparan sulfate, to promote the aggregation of amyloidogenic proteins and to bind to the resulting aggregates. Preformed amyloid fibrils of human muscle acylphosphatase and human lysozyme, proteins with a net positive charge at physiological pH values, were found to bind tightly to the negatively charged DNA or ATP. The effects of the polyelectrolytes on the kinetics of aggregation were studied for acylphosphatase, and the presence of ATP, DNA, or heparin was found to increase its aggregation rate dramatically, with a degree dependent on the net charge and size of the polyanion. Magnesium or calcium ions were found to attenuate, and ultimately to suppress, these interactions, suggesting that they are electrostatic in nature. Moreover, heparin was found to stabilize the aggregated state of acylphosphatase through compensation of electrostatic repulsion. Noteworthy, differences in affinity between native and aggregated acylphosphatase with heparin suggest that amyloid fibrils can themselves behave as polyelectrolytes, interacting very strongly with other polyelectrolytes bearing the opposite charge. Within an in vivo context, the strengthening of the electrostatic interactions with other biological polyelectrolytes, as a consequence of protein misfolding and aggregation, could therefore result in depletion of essential molecular components and contribute to the known cytotoxicity of amyloid fibrils and their precursors.

Amyloid↗
PubMed2006

Recombinant amyloidogenic domain of ApoA-I: analysis of its fibrillogenic potential.

A variety of amyloid diseases are associated with fibrillar aggregates from N-terminal fragments of ApoA-I generated through a largely unexplored multi-step process. The understanding of the molecular mechanism is impaired by the lack of suitable amounts of the fibrillogenic polypeptides that could not be produced by recombinant methods so far. We report the production and the conformational analysis of recombinant ApoA-I 1-93 fragment. Similarly to the polypeptide isolated ex vivo, a pH switch from 7 to 4 induces a fast and reversible conformational transition to a helical state and leads to the identification of a key intermediate in the fibrillogenesis process. Limited proteolysis experiments suggested that the C-terminal region is involved in helix formation. The recombinant polypeptide generates fibrils at pH 4 on a time scale comparable with that of the native fragment. These findings open the way to studies on structural, thermodynamic, and kinetic aspects of ApoA-I fibrillogenesis.

Amyloid↗
PubMed2006

Characterization of the nanoscale properties of individual amyloid fibrils.

We report the detailed mechanical characterization of individual amyloid fibrils by atomic force microscopy and spectroscopy. These self-assembling materials, formed here from the protein insulin, were shown to have a strength of 0.6 +/- 0.4 GPa, comparable to that of steel (0.6-1.8 GPa), and a mechanical stiffness, as measured by Young's modulus, of 3.3 +/- 0.4 GPa, comparable to that of silk (1-10 GPa). The values of these parameters reveal that the fibrils possess properties that make these structures highly attractive for future technological applications. In addition, analysis of the solution-state growth kinetics indicated a breakage rate constant of 1.7 +/- 1.3 x 10(-8) s(-1), which reveals that a fibril 10 mum in length breaks spontaneously on average every 47 min, suggesting that internal fracturing is likely to be of fundamental importance in the proliferation of amyloid fibrils and therefore for understanding the progression of their associated pathogenic disorders.

Amyloid↗
PubMed2006

X-ray scattering study of the effect of hydration on the cross-beta structure of amyloid fibrils.

We have investigated the effect of sample hydration on the wide-angle X-ray scattering patterns of amyloid fibrils from two different sources, hen egg white lysozyme (HEWL) and an 11-residue peptide taken from the sequence of transthyretin (TTR105-115). Both samples show an inter-strand reflection at 4.7 A and an inter-sheet reflection which occurs at 8.8 and approximately 10 A for TTR105-115 and HEWL fibrils, respectively. The positions, widths, and relative intensities of these reflections are conserved in patterns obtained from dried stalks and hydrated samples over a range of fibril concentrations. In 2D scattering patterns obtained from flow-aligned hydrated samples, the inter-strand and inter-sheet reflections showed, respectively, axial and equatorial alignment relative to the fibril axis, characteristic of the cross-beta structure. Our results show that the cross-beta structure of the fibrils is not a product of the dehydrating conditions typically employed to produce aligned samples, but is conserved in individual fibrils in hydrated samples under dilute conditions comparable to those associated with other biophysical and spectroscopic techniques. This suggests a structure consisting of a stack of two or more sheets whose interfaces are inaccessible to bulk water.

Amyloid↗
PubMed2006

Prefibrillar amyloid aggregates could be generic toxins in higher organisms.

More than 40 human diseases are associated with fibrillar deposits of specific peptides or proteins in tissue. Amyloid fibrils, or their precursors, can be highly toxic to cells, suggesting their key role in disease pathogenesis. Proteins not associated with any disease are able to form oligomers and amyloid assemblies in vitro displaying structures and cytotoxicity comparable with those of aggregates of disease-related polypeptides. In isolated cells, such toxicity has been shown to result from increased membrane permeability with disruption of ion homeostasis and oxidative stress. Here we microinjected into the nucleus basalis magnocellularis of rat brains aggregates of an Src homology 3 domain and the N-terminal domain of the prokaryotic HypF, neither of which is associated with amyloid disease. Prefibrillar aggregates of both proteins, but not their mature fibrils or soluble monomers, impaired cholinergic neuron viability in a dose-dependent manner similar to that seen in cell cultures. Contrary to the situation with cultured cells, however, under our experimental conditions, cell stress in tissue is not followed by a comparable level of cell death, a result that is very likely to reflect the presence of protective mechanisms reducing aggregate toxicity. These findings support the hypothesis that neurodegenerative disorders result primarily from a generic cell dysfunction caused by early misfolded species in the aggregation process.

Amyloid↗
PubMed2006

Protein unfolding, amyloid fibril formation and configurational energy landscapes under high pressure conditions.

High hydrostatic pressure induces conformational changes in proteins ranging from compression of the molecules to loss of native structure. In this tutorial review we describe how the interplay between the volume change and the compressibility leads to pressure-induced unfolding of proteins and dissociation of amyloid fibrils. We also discuss the effect of pressure on protein folding and free energy landscapes. From a molecular viewpoint, pressure effects can be rationalised in terms of packing and hydration of proteins.

Amyloid↗
PubMed2006

Identification of the core structure of lysozyme amyloid fibrils by proteolysis.

Human lysozyme variants form amyloid fibrils in individuals suffering from a familial non-neuropathic systemic amyloidosis. In vitro, wild-type human and hen lysozyme, and the amyloidogenic mutants can be induced to form amyloid fibrils when incubated under appropriate conditions. In this study, fibrils of wild-type human lysozyme formed at low pH have been analyzed by a combination of limited proteolysis and Fourier-transform infrared (FTIR) spectroscopy, in order to map conformational features of the 130 residue chain of lysozyme when embedded in the amyloid aggregates. After digestion with pepsin at low pH, the lysozyme fibrils were found to be composed primarily of N and C-terminally truncated protein species encompassing residues 26-123 and 32-108, although a significant minority of molecules was found to be completely resistant to proteolysis under these conditions. FTIR spectra provide evidence that lysozyme fibrils contain extensive beta-sheet structure and a substantial element of non beta-sheet or random structure that is reduced significantly in the fibrils after digestion. The sequence 32-108 includes the beta-sheet and helix C of the native protein, previously found to be prone to unfold locally in human lysozyme and its pathogenic variants. Moreover, this core structure of the lysozyme fibrils encompasses the highly aggregation-prone region of the sequence recently identified in hen lysozyme. The present proteolytic data indicate that the region of the lysozyme molecule that unfolds and aggregates most readily corresponds to the most highly protease-resistant and thus highly structured region of the majority of mature amyloid fibrils. Overall, the data show that amyloid formation does not require the participation of the entire lysozyme chain. The majority of amyloid fibrils formed from lysozyme under the conditions used here contain a core structure involving some 50% of the polypeptide chain that is flanked by proteolytically accessible N and C-terminal regions.

Amino Acid Sequence↗
PubMed2006

Spatial persistence of angular correlations in amyloid fibrils.

Using atomic force microscopy height maps, we resolve and quantify torsional fluctuations in one-dimensional amyloid fibril aggregates self-assembled from three different representative polypeptide systems. Furthermore, we show that angular correlation in these nanoscale structures is maintained over several microns, corresponding to many thousands of molecules along the fibril axis. We model disorder in the fibril in respect of both thermal fluctuations and structural defects, and determine quantitative values for the defect density, as well as the energy scales involved in the fundamental interactions stabilizing these generic structures.

Amyloid↗
PubMed2006

Apomyoglobin reveals a random-nucleation mechanism in amyloid protofibril formation.

Protofibrils (PFs) represent the earliest fibrillar species that occur in the course of amyloid fibril formation. Using apomyoglobin, we report here that PFs arise from a multi-step reaction and that they are preceded by an ensemble of non-fibrillar particles (NFPs). These intermediate aggregates encompass nascent elements of amyloid structure and can act as seeds in PF formation. Taken together with the observation that PFs often protrude from NFPs, our data suggest that PFs form by a random nucleation mechanism in which the polypeptide chains sample many different aggregated conformations. Once the appropriate structural characteristics are acquired, PFs are formed by addition of further polypeptide chains.

Amyloid↗
PubMed2006

The component polypeptide chains of bovine insulin nucleate or inhibit aggregation of the parent protein in a conformation-dependent manner.

Amyloid fibrils are typically rigid, unbranched structures with diameters of approximately 10 nm and lengths up to several micrometres, and are associated with more than 20 diseases including Alzheimer's disease and type II diabetes. Insulin is a small, predominantly alpha-helical protein consisting of 51 residues in two disulfide-linked polypeptide chains that readily assembles into amyloid fibrils under conditions of low pH and elevated temperature. We demonstrate here that both the A-chain and the B-chain of insulin are capable of forming amyloid fibrils in isolation under similar conditions, with fibrillar morphologies that differ from those composed of intact insulin. Both the A-chain and B-chain fibrils were found to be able to cross-seed the fibrillization of the parent protein, although these reactions were substantially less efficient than self-seeding with fibrils composed of full-length insulin. In both cases, the cross-seeded fibrils were morphologically distinct from the seeding material, but shared common characteristics with typical insulin fibrils, including a very similar helical repeat. The broader distribution of heights of the cross-seeded fibrils compared to typical insulin fibrils, however, indicates that their underlying protofilament hierarchy may be subtly different. In addition, and remarkably in view of this seeding behavior, the soluble forms of the A-chain and B-chain peptides were found to be capable of inhibiting insulin fibril formation. Studies using mass spectrometry suggest that this behavior might be attributable to complex formation between insulin and the A-chain and B-chain peptides. The finding that the same chemical form of a polypeptide chain in different physical states can either stimulate or inhibit the conversion of a protein into amyloid fibrils sheds new light on the mechanisms underlying fibril formation, fibril strain propagation and amyloid disease initiation and progression.

Amyloid↗
PubMed2006

Folding and fibril formation of the cell cycle protein Cks1.

The Saccharomyces cerevisiae Cks protein Cks1 has a COOH-terminal glutamine-rich sequence not present in other homologues. Cks proteins domain swap to form dimers but unique to Cks1 is the anti-parallel arrangement of protomers within the dimer. Despite the differences in Cks1 compared with other Cks proteins, we find the domain swapping properties are very similar. However, aggregation of Cks1 occurs by a route distinct from the other Cks proteins studied to date. Cks1 formed fibrillar aggregates at room temperature and neutral pH. During this process, Cks1 underwent proteolytic cleavage at a trypsin-like site into two fragments, the globular Cks domain and the glutamine-rich COOH terminus. At high protein concentrations, the rate of fibril formation was the same as the rate of proteolysis. The dominant species present within the fibrils was the glutamine-rich sequence. Consistent with this result, fibril formation was enhanced by addition of trypsin. Moreover, a truncated variant lacking the glutamine-rich sequence did not form fibrils under the same conditions. A lag phase at low protein concentrations indicates that fibril formation occurs through a nucleation and growth mechanism. The aggregates appear to resemble amyloid fibrils, in that they show the typical cross-beta x-ray diffraction pattern. Moreover, infrared spectroscopy data indicate that the glutamine side chains are hydrogen-bonded along the axis of the fibril. Our results indicate that the proteolytic reaction is the crucial step initiating aggregation and demonstrate that Cks1 is a simple, tunable model system for exploring aggregation mechanisms associated with polyglutamine deposition diseases.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Expanding to fill the gap: a possible role for inert biopolymers in regulating the extent of the 'macromolecular crowding' effect.

We discuss the potential for inert biopolymers existing in cells to play a role in regulating the macromolecular crowding effect via their ability to undergo shape changing structural transitions. We have explored this possibility by the use of theory and experiment. The theoretical component utilized Monte-Carlo based simulations to examine the folding of a hypothetical protein in a concentrated environment of hard spheres which are themselves capable of reversible expansion and contraction. The experimental component of the study involved examination of the effect of different sized crowding agents on the thermally induced denaturation of cytochrome c [in phosphate buffered saline solution containing 1.0M guanidinium hydrochloride at pH 7.0]. On the basis of our findings we suggest that in a crowded solution environment the presence of a non-reactive polymer capable of reversible expansion/contraction via folding and unfolding may alter the excluded volume component of the solution. This ability would confer on the non-reactive polymer a novel role in influencing other processes in solution affected by macromolecular crowding.

Animals↗
PubMed2006

Cytochrome display on amyloid fibrils.

Protein amyloid fibrils can be functionalized by coating the core protofilament with high concentrations of proteins and enzymes. This can be done elegantly by appending a functional domain to an amyloidogenic protein monomer, then assembling the monomers into a fibril. To display an array of biologically functional porphyrins on the surface of protein fibrils, we have fused the sequence of the small, soluble cytochrome b562 to an SH3 dimer sequence that can form classical amyloid fibrils rapidly under well-defined conditions. The resulting fusion protein also forms amyloid fibrils and, in addition, binds metalloporphyrins, at half of the porphyrin binding sites as shown by UV-vis and NMR spectroscopies. Once metalloporphyrins are bound to the fibrils, the resulting holo-cytochrome domains are spectroscopically identical to the wild type cytochrome. The concentration of metalloporphyrins on a saturated fibril is estimated to be of the order of approximately 20 mM, suggesting that they could be interesting systems for applications in nanotechnology.

Amyloid↗
PubMed2006

Probing the mechanism of amyloidogenesis through a tandem repeat of the PI3-SH3 domain suggests a generic model for protein aggregation and fibril formation.

Aggregation of the SH3 domain of the PI3 kinase, both as a single domain and as a tandem repeat in which the C terminus of one domain is linked to the N terminus of another by a flexible linker of ten glycine/serine residues, has been studied under a range of conditions in order to investigate the mechanism of protein aggregation and amyloid formation. The tandem repeat was found to form amyloid fibrils much more readily than the single domain under the acidic conditions used here, and the fibrils themselves have higher morphological homogeneity. The folding-unfolding transition of the PI3-SH3 domain shows two-state behaviour and is pH dependent; at pH 3.6, which is near the pH mid-point for folding and only slightly below the isoelectric point of the protein, both the single domain and the tandem repeat spontaneously form broad distributions of soluble oligomers without requirement for nucleation. Under prolonged incubation under these conditions, the oligomers convert into thin, curly fibrils that interact with thioflavin-T, suggesting that they contain an organised beta-sheet structure. Under more acidic conditions (pH 2.0) where the proteins are fully denatured and carry a positive net charge, long, straight fibrils are formed in a process having a pronounced lag phase. The latter was found to be reduced dramatically by the addition of oligomers exceeding a critical size of approximately 20 molecules. The results suggest that the process of aggregation of these SH3 domains can take place by a variety of mechanisms, ranging from downhill formation of relatively amorphous species to nucleated formation of highly organised structures, the relative importance of which varies greatly with solution conditions. Comparison with the behaviour of other amyloidogenic systems suggests that the general mechanistic features outlined here are likely to be common to at least a wide variety of peptides and proteins.

Amyloid↗
PubMed2006

Normal and aberrant biological self-assembly: Insights from studies of human lysozyme and its amyloidogenic variants.

Studies of lysozyme have played a major role over several decades in defining the general principles underlying protein structure, folding, and stability. Following the discovery some 10 years ago that two mutational variants of lysozyme are associated with systemic amyloidosis, these studies have been extended to investigate the mechanism of amyloid fibril formation. This Account describes our present knowledge of lysozyme folding and misfolding, and how the latter can give rise to amyloid disease. It also discusses the significance of these studies for our general understanding of normal and aberrant protein folding in the context of human health and disease.

Amyloid↗